“I wish I were a fish,” Henry Limpet whispers on a Coney Island pier as he reflects on life’s disappointments in the movie The Incredible Mr. Limpet.

Moments later, there’s a splash and all that remains of Henry is his hat, bobbing on the waves.

Deeper and deeper, Henry descends, slowly switching limbs for fins until all that remains is a near-sighted fish wearing horned-rim glasses.

The option of solving dilemmas and finding purpose in life by transforming completely into a fish may only be open to comedic actor Don Knotts and other characters in Hollywood films, but for individual patients struggling with disease, hope for diagnosis and personalized treatment may soon lie in the transformation of fish, worms and other organisms into models of the patient’s condition.

More than mammals

Despite being very distant on the evolutionary timeline, non-mammalian models can still provide significant insights into human disease and the impact of potential therapeutics, as suggested by Baylor College of Medicine’s Hugo Bellen and colleagues in a recent review.

“Orthologous genes often cause different phenotypes in different species, yet the proteins encoded by these genes have similar molecular functions,” they wrote. “To discover phenologs, one can cluster genes known to cause a human disease and determine the phenotypes caused by mutations in the corresponding genes in model organisms.”

The authors offered the example of human breast cancer genes related to genes in the nematode Caenorhabditis elegans (C. elegans) that mutate to cause a high frequency of male offspring. Although the phenotypes do not appear related, they suggested, the outcomes derive from common underlying genetic defects, such as DNA damage response.

“For genes that are evolutionarily conserved, simple model organisms with short generation times and amenable to inexpensive and efficient genetic manipulations can yield rapid insights into basic biological functions through detailed in-vivo studies,” the authors continued.

“Finally, when using one species to understand another, a key experiment is to determine how interchangeable genes are between species if one wishes to assess whether a human variant may be pathogenic.”

The worm turns

As suggested above, one organism that has seen increasing use in disease modeling is C. elegans, a small transparent worm that—alongside Drosophila melanogaster (fruit fly)—might be the most thoroughly studied and best understood invertebrate, if not animal, in the world. Every cell in the mature organism and its lineage from fertilized ovum to adult has been mapped, making changes in morphology that much more obvious.

“That is part of the reason why the full molecular biology tool kit can be leveraged on that animal,” says Matt Beaudet, CEO of NemaMetrix, a company focused on leveraging C. elegans as a model organism. “But on the phenotyping side, it also means that it is so well understood that if even an extra cell pops up, you know that it is of significance. And when it comes to looking at the impact of drugs on the biology of a real animal, that sort of resolution of phenotyping becomes important.”

“There is nothing that can compare to C. elegans for how agreeable they are to genetic manipulation and phenotyping, and figuring out the impact that the gene has on the actual core biology,” he enthuses.

In November, with an eye to further enable the translation of worm genes to morphology and behavior, NemaMetrix announced its merger with commercial partner Knudra Transgenics. As Beaudet explains, both companies had been focused on the translation but approached the challenge from different yet complementary directions: Knudra focused on the generation of transgenic animals and NemaMetrix focused on behavior and phenotyping, particularly through its ScreenChip platform.

“Both of us were looking at how we can translate what we understand about the genome into something that’s actionable,” Beaudet adds. “And that mission led us to C. elegans.”

Echoing Bellen, Beaudet suggests that despite their significant differences morphologically, C. elegans and humans show a high degree of conservation in terms of their core machinery. This conservation is reflected genetically. Within the 7,000 human genes known to be involved in disease, the worm has about 80 percent homology.

In a recent review, Washington University’s Holly Kinser and Zachary Pincus offered their thoughts on what made C. elegans an attractive organism for a variety of studies, including their own interest in neurobiology.

“C. elegans offers the utility of a whole-animal model with the simplicity and convenience of single cells: the animal has well-defined tissues, distinct organ systems and exhibits a variety of complex behaviors, yet its small size, short reproductive cycle and ability to self-fertilize make it simple and inexpensive to maintain on both solid and liquid media,” they wrote.

“The connections between the animal’s 302 neurons have been mapped, and its transparent body is conducive to calcium imaging, optogenetics and other fluorescence-based techniques, allowing for observation of both single neurons and the entire nervous system.”

And because its food source is bacteria, C. elegans has become a valuable resource in the discovery and development of new antibiotics, anti-virulence factors and immunomodulators.

Getting personal

But as Beaudet explains, the advent of technologies that permit researchers to swap worm genes for human genes—for example, CRISPR, TALENS and ZFNs—has changed the landscape of what is possible with the worm.

Earlier this year, David Bulger and colleagues at Cambridge University and the National Institute of Diabetes and Digestive and Kidney Diseases developed phenotypic assays in C. elegans of mutations of the DAF-2 insulin-like receptor gene. They then used CRISPR to confirm their findings in a subset of worm mutations, as well as inserting a series of human disease mutations.

“Our results were a mix of the expected and unexpected, highlighting the importance of testing allele function in the context of a whole-animal system in which receptor processing and function are integrated with the entire metabolic program,” the authors wrote. “Our experiences with the insulin-like signaling pathway in C. elegans demonstrate the power of this model system to better understand the possible organismal-level consequence of mutations in conserved physiological processes.”

Beaudet highlights the importance of rapid gene editing for looking at multiple variants of a disease, offering the example of BRCA1/BRCA2 breast cancer genes and their approximately 4,000 variants.

“We can actually create all 4,000 variants, if needed,” he suggests. “That way you can look at the impact that a drug is having, not just on a representative variant, but on all the variants that are present in a population.”

Chris Hopkins, Knudra founder and now chief scientific officer of the combined companies, describes this as a personalized medicine approach.

“If a patient comes in with a novel variant, you can install it [in the worm] and study them,” he explains.

Such an approach was exemplified by Anne Hart and colleagues at Brown University last year when a patient was admitted to the hospital with unclassifiable ataxia-like symptoms. Exome sequencing of the patient revealed a G316S mutation in ATP1A3, a subunit of an enzyme involved in the maintenance of membrane potential.

To determine whether this mutation was linked to the patient’s symptoms, the researchers used CRISPR to insert the human mutation into the C. elegans homologue EAT-6. They then monitored the impact of the mutation in the worm using behavioral assays.

Interestingly, whereas common EAT-6 mutations are recessive—only homozygotes experience problems—the human G-to-S mutation was dominant, as both hetero- and homozygotic worms were debilitated, consistent with the human patient, who was heterozygous.

“These results indicate that the mutation is dominant and impairs the neuromuscular function,” the authors wrote. “Thus, we conclude that the de-novo G316S mutation in ATP1A3 likely causes or contributes to patient symptoms.

“More broadly, we conclude that for conserved genes, it is possible to rapidly and easily model human diseases in C. elegans using CRISPR/Cas9 genome editing.”

Hopkins also sees the potential for researchers to understand the possible roles of background mutations, such as whether they enhance or suppress disease phenotypes.

“The ability now to build all these models and make all these crosses where we’re building multigenic conditions in a pseudo-personalized way is there in mice, but the timeline is long,” he presses. “You’re talking six months to a year. In C. elegans, it takes two animals maybe a couple of weeks and you’re looking at data in under a month.”

“The older approach to drug discovery, of one drug that’s going to impact one disease that’s prevalent in a large portion of the population, is changing,” Beaudet adds. “And the definitions of diseases are becoming narrower and narrower.”

“In a disease like cardiac arrhythmia, there are multiple genetic components that lead to the same set of symptoms,” he continues. “It opens up the question of should there be five different medicines that can be treated based on the five different genetic variants.”

Effectively, Hopkins says, as each of these diseases splinters into personalized variants, we are heading to a point where all disease is rare disease.

The translation of those genetic modifications into interpretable and actionable data is where the company’s ScreenChip platform comes in.

As Kat McCormick, NemaMetrix’s director of R&D, explains, the system was initially designed to perform specific electrical measurements that were very difficult and required specialized skills and equipment.

“What we did with the ScreenChip system was allow researchers to take those neuromuscular phenotyping assays and make them accessible,” she says. “And then on top of that core technology, we can add in some of the more visible phenotypes that C. elegans also display, and also bring in the genetic tools so that we can work out what happens when you disrupt the gene function.”

Alongside the screening platform, NemaMetrix has also been developing a series of fluorescent staining kits designed to further enhance worm phenotyping experiments.

Said Beaudet at the June launch of the RediStain fluorescent kits: “The introduction of fluorescent staining kits to our ScreenChip System will make previously invisible phenotypes easy to visualize, allowing scientists to generate more and better quality data on neuronal and physiological responses to a wide range of genetic and environmental changes.”

Other groups are meanwhile looking to evolve the panoply of fluorescent proteins that can be introduced to the worm (or other organisms) for expression in specific cell types or under specific regulation to monitor the impact of disease genes or potential therapies on morphology and cell migration.

Earlier this year, for example, Dominique Glauser and colleagues at University of Fribourg and Cambridge’s MRC Laboratory of Molecular Biology used C. elegans to compare the in-vivo activity of green fluorescent protein (GFP) with mNeonGreen, a new derivative of a yellow fluorescent protein. In vitro, mNeonGreen was found to be significantly brighter than GFP, offering hope that the new marker might be useful in studies where gene expression is particularly low.

The researchers noted that across a variety of adult worm tissues, mNeonGreen was significantly brighter than GFP, whether it was expressed alone or as part of a protein fusion. As well, the researchers could target mNeonGreen to specific subcellular regions and use it to follow protein trafficking.

“With the growing application of efficient single-copy transgene integration methods and the boom in genome-editing techniques that enable the addition of fluorescent tags to endogenous coding sequences, the development of brighter fluorescent tags will fulfill an important emerging need,” the researchers wrote. “Stronger signal can ease expression pattern definition, as illustrated with our reporter approach in low-expression genes, and simplify protein tracking within cells in vivo.”

“Furthermore, we anticipate it could improve the visualization of small cellular structures and facilitate high-throughput experiments where time is constraining, or when fluorescence detection sensitivity is limiting.”

Combined, these technological advances offer researchers the opportunity to try experiments that might not otherwise make sense, says Beaudet, potentially opening the door to serendipitous findings.

“When you have that license to play, that lower barrier to try out live animal testing, we’ve found that it opens up new possibilities such as reverse drug screens where you have a couple of lead compounds and you want to screen if there is any secondary disease that they have an impact on, positively or negatively,” he enthuses. “You can screen against diseases that, on paper, would just make no sense. But, because the barrier is so low, you can just try it out. And that is something you cannot do in mice.”

Despite all of this success in worms, Beaudet is quick to note that C. elegans isn’t a replacement for other model organisms.

“They all need to work together,” he says. “It is really looking at what is the strength of the C. elegans animal, which is speed, cost and accessibility, essentially because it is so well understood.”

“The limitations of it definitely are the advantages of mice: mice have hearts, mice have kidneys and mice have livers,” he offers. “Mice have two genders and live births, and a bunch of other things that you would want to see how a disease progresses and a drug impacts that disease in that larger mammalian system.”

“Our goal has always been to see how quickly we can get somebody to have the confidence in their data to move on to the next animal,” he continues. “If you’re able to eliminate drugs that are toxic or have a side impact on a separate disease quickly, or if you’re able to—because it costs almost nothing to try out new drugs—you’re able to screen libraries that are 10,000 or 100,000 strong, and find the lead compounds quicker and then move on to the clinical trial, that would be great.”

That said, the shift from invertebrate models doesn’t necessarily mean heading directly to mice and other mammals.

Going fishing

Like C. elegans, the molecular resources to substitute human genes for their animal homologues has led to a rapid expansion in the use of fish as models of human disease pathology, and particularly zebrafish (Danio rerio).

“I would say more than 70 percent genes including most druggable targets are conserved between zebrafish and humans,” explains Xiao-Yan Wen, director of the Zebrafish Centre for Advanced Drug Discovery (ZCADD) at Toronto’s St. Michael’s Hospital.

“The basic organ development, physiology and most molecular pathways are also conserved,” he continues, “so fish can be used to model different diseases such as heart disease, neural disease, cancer, diabetes, inflammation and many more.”

As Wen explains, he established ZCADD—Canada’s first and only robotic zebrafish high-throughput facility—to fully explore the potential of using zebrafish for drug screening.

The facility relies on liquid handlers to dispense embryos into 96-well plates, as well as candidate compounds for screening experiments, and a confocal plate scanner with 3D imaging capability that allows the researchers to monitor morphological changes or other markers of compound efficacy or toxicity, separately or simultaneously.

“The fish community already has lots of transgenic lines with fluorescently labeled cell types or organs,” Wen explains. “Using them, we can watch organ formation, and in some cases, directly watch drug efficacy in vivo, which is impossible in mouse.”

The center is also equipped with devices to study behavior in adult and larval zebrafish, providing a high-throughput screen for endpoints such as changes in locomotion.

Earlier this year, Wen and colleagues described what they believed to be the key advantages of zebrafish as a prototype for modeling human disease.

“With a sequenced genome, the zebrafish offers a unique and robust prototype for disease mod­eling,” they said, because of:

Its remarkable physiological similarity and degree of functional conservation in basic cell-biological processes to humans;

The relative ease of embryonic manipulation and whole-body imaging;

High fecundity;

Low cost;

Transparency; and

The availability of various molec­ular tools for forward-reverse genetics and genome editing.

Sharing Wen’s enthusiasm for zebrafish are Vanderbilt University School of Medicine’s Charles Williams and Charles Hong.

“Once a disease phenotype or a surrogate phenotype is established in zebrafish mutants, a therapeutic screen for compounds that ameliorate these phenotypes should be straightforward,” they wrote in a recent overview. “With the advances in genomic sequencing technologies, the number of ultra-rare genetic diseases is expected to increase significantly in the coming decade.”

As Wen explains, zebrafish offer many different ways to create disease models.

“You can use the chemical method, such as putting a neurotoxin MPP+ in the water to damage the dopaminergic neuron to build model for Parkinson’s disease,” he suggests. “You can also use the physical method, such as cutting the tail fin to generate a wound-healing model to study neutrophil migration and the inflammation processes.”

A good example of the chemical method was in the development of a model for sepsis that Wen and colleagues described earlier this year, where they immersed larval zebrafish in solutions of lipopolysaccharide (LPS).

Using microangiography with agents such as quantum dots and FITC-dextran, the researchers noted clear signs of vascular leakage. Using RT-PCR, they also noted diminished expression of cellular junction proteins.

The researchers screened LPS-treated larvae against a library of compounds, looking for anything that might ameliorate or prevent LPS damage, as signalled by edema, radical oxygen species (ROS) generation or mortality. Of the 96 compounds screened, 10 were shown to rescue all three phenotypes and one of those three—fasudil—was known to rescue LPS-induced vascular leakage in mice, validating the zebrafish model.

The results highlight the opportunities for drug discovery based on phenotype screening rather than the more traditional target-based approach.

“Phenotype-based screening in whole organisms allows for testing of novel compounds while evaluating complex biological processes at the whole-organism level in an unsupervised and systematic fashion, enhancing the potential to detect more plausible targets for both preclinical and mechanistic studies,” the authors suggested.

As discussed earlier, however, perhaps the greatest area of growth in recent years has been in the development of genetic models, whether through the over-expression of human disease genes via transgenics, knocking out the fish gene function using CRISPR or knocking down gene expression using morpholino oligonucleotides.

Earlier this year, Saadet Mercimek-Andrews of Toronto’s Hospital for Sick Children and colleagues including Wen used CRISPR-Cas9 technology to generate a zebrafish model of pyroxidine-dependent epilepsy (PDE), knocking out ALDH7A1, a gene involved in lysine catabolism.

“Our knock-out ALDH7A1 zebrafish model showed spontaneous rapid increase in locomotion and a rapid circling swim behavior followed by loss of posture as seizure-like locomotor behavior as early as [eight days post-fertilization], which resulted in death shortly after this seizure-like locomotor behavior,” the researchers described.

Furthermore, EEG recordings of embryos exhibiting seizure-like locomotor behavior demonstrated large amplitude spike discharges that could not be ameliorated with antiseizure medications, but were normalized with pyroxidine treatment. They were also able to detect build-up of various metabolic markers characteristic of PDE; this is the first time this has been possible in a model organism, according to the authors.

“Our model will allow us to perform moderate-to-high throughput drug screening to assess effectiveness of drugs not only on seizure-like locomotor behavior and survival into adulthood, but also normalization of alpha-AASA, P6C and PA accumulation,” the researchers wrote. “Our knockout model will help us to identify the most effective compound to be able to treat human PDE-ALDH7A1.”

This research highlights one area in which Wen believes zebrafish can be particularly useful: disease gene discovery and validation.

There are lots of genetic screens in humans to hunt for novel disease genes, he says, but a huge challenge is how to effectively validate them. Zebrafish models can be used to quickly validate a gene’s function and define disease-causing mutations.

Another example of this is Wen’s efforts with University of British Columbia’s Clara Van Karnebeek, Lausanne University Hospital’s Andrea Superti-Furga and others to validate a new disease gene called NANS, a sialic acid synthase that is also involved in skeletal and mental development. Zebrafish knock-outs of NANS resulted in fish with abnormal skeletal development that could be partially rescued by the addition of exogenous sialic acid, thus offering a screening platform for drugs to treat the disease.

Which leads us to the second area in which Wen sees potential for zebrafish: drug discovery.

Screening phenotypes

“As a phenotype-based in-vivo screen, zebrafish has excellent target engagement to screen for the entire pathway, very different from the traditional single target-based screens,” Wen continues. “With zebrafish, you can screen for both drugs as well as drug metabolites.”

Again, Wen’s enthusiasm was echoed by Williams and Hong.

“A phenotypic screen, by definition, identifies chemotypes that affect a biologically meaningful target or targets, including key nodes responsible for integrating cell pathways and behaviors,” they wrote. “Importantly, since a phenotypic screen is conducted without regard to a priori knowledge of targets, it has the potential to discover new therapeutic targets, which may have greater impact at the systems level than established targets. Moreover, in contrast to target-based screens, a phenotypic screen permits discovery of compounds that affect a desired outcome via engaging multiple targets in a synergistic manner that may not have been otherwise anticipated.”

Highlighting this impact was work by Université de Montréal’s Pierre Drapeau and Alex Parker, who, with Wen and others, recently described a high-throughput screen of clinically approved small-molecule libraries in amyotrophic lateral sclerosis (ALS). The study also highlighted the power of using multiple model species.

In an initial screen of 3850 compounds in a C. elegans ALS model, the researchers identified 24 compounds that rescued worm paralysis, of which 13 were found to belong to a family of neuroleptics. The researchers then validated these hits in a zebrafish model of ALS, confirming 10 compounds that rescued fish mobility.

Of these 10, they pursued the most potent lead compound—pimozide, an FDA-approved compound for the treatment of chronic psychosis, Tourette syndrome and resistant tics.

Although pimozide was initially designed as a dopaminergic D2 antagonist, it has also been shown to block T-type Ca2+ currents, an activity the researchers confirmed in both C. elegans and zebrafish. Finding the compound was able to restore neuromuscular transmission in both of these models, as well as in a mouse model of ALS, the researchers moved pimozide into a small clinical trial of subjects with ALS, where it showed promise.

“For us, this is an indication that we found the right therapeutic target,” said Drapeau in announcing the findings. “Pimozide acts directly on the neuromuscular junction, as shown in our animal models. We don’t yet know whether pimozide has a curative effect, or whether it only preserves normal neuromuscular function to at least stabilize the disease.”

A larger clinical study initiated recruitment in November.

“In our study, we combined the use of several model systems (C. elegans, zebrafish and mice) and have capitalized on the strength of each organism to identify small-molecule therapeutics for ALS,” the researchers concluded. “Our findings demonstrate that simple animal models can be valuable in the preclinical pipeline to bridge the gap between in-vitro assays and more costly screens in mammals.

“More importantly, the effect of pimozide in our C. elegans and zebrafish models of ALS and humans suggest not only that simple animal models are useful in identifying compounds that hold promise for the treatment of ALS, but they may be accurate predictors of clinical trial outcomes.”

Although Wen works extensively with academic researchers, he is very keen to expand his partnerships with the pharmaceutical sector, highlighting his efforts with a new partnership with Janssen Pharmaceuticals, part of his CAN-BIND (Canadian Biomarker Integration Network for Depression) collaboration with University Health Network’s Sidney Kennedy.

For this project, Wen’s team received pilot funding to build fluorescent zebrafish reporters to study microRNA biomarkers in depression. These biomarkers were first identified from CAN-BIND clinical program in depression patients.

“[The] zebrafish not only allows us to do cool science, comparing to the traditional mouse models, it also provides us a unique tool, bridging invertebrates to human for cheaper and quicker research translation and commercialization,” he says.

Wen is now very interested in screening natural compounds, including marine-derived compounds and traditional herbal medicines. He is developing partnerships in China to study the Chinese herbal medicines with zebrafish—an area, he argues, that has been largely unexplored.

Wen is also actively trying to build the zebrafish community, organizing a series of international meetings known as Zebrafish for Personalized/Precision Medicine Conference. Held in Toronto every two years, the most recent conference took place in September and brought about 200 zebrafish researchers and clinicians to discuss everything from new disease models to drug screening technologies to correlations of model pathology with human electronic health records.

Seems that John Merrick’s cries in The Elephant Man that “I am not an animal” may have been a little premature.

This past fall, the In Vivo Pharmacology division at The Jackson Laboratory (JAX) announced the launch of a collection of new human acute myeloid leukemia (AML) preclinical mouse models that JAX officials say more broadly represent AML subtypes seen in the human population. These early passaged tumor models, available and characterized in NSG-SGM3 mice, were donated by Dr. David Weinstock’s lab at the Dana-Farber Cancer Institute to JAX to improve the accessibility of AML models and propel scientific discoveries globally.

“The diversity represented by this panel allows researchers to better hone in on specific AML targets, creating an invaluable tool set to support drug discovery,” said Weinstock.

“This is an important milestone in The Jackson Laboratory’s and Dana-Farber’s longstanding missions to improve accessibility of tumor models and support research and drug discovery,” said Dr. Walter Ausserer, associate general manager of In Vivo Services at JAX. “To that end, we have focused on making this panel highly accessible to the research community and can supply cohorts of these AML-bearing mice to scientists in both academic and industrial settings, in addition to executing drug efficacy studies at our Sacramento-based In Vivo pharmacology laboratory.”

Meanwhile, fall also saw a bit of activity from Crown Bioscience promoting its FATZO model as a significantly improved rodent model for obesity, dysmetabolism and type 2 diabetes in preclinical studies. According to Crown, this model has been designed to present with polygenic obesity and a metabolic pattern of hyperglycemia and hyperinsulinemia.

“The FATZO mouse is an improved translatable model that provides us with a better understanding of the physiological and cellular mechanisms that lead from obesity, metabolic disorders, to diabetes,” said Charles Van Jackson, chief scientific officer of CrownBio Indiana. “The data presented in these papers demonstrates that FATZO has several advantages over traditional rodent models and will impact the speed of drug discovery.”

Soon thereafter, Crown also released news of the availability of the proprietary FATZO model in a European-based laboratory, noting that “The establishment of this improved translational model provides the European scientific community with unfettered access to an advanced research tool for use in preclinical studies of obesity, dysmetabolism and type 2 diabetes.”

And, in late November, Crown also made note of the fact that the FATZO model had been used in recent preclinical work conducted by Eli Lilly and Co. for its type 2 diabetes research programs.

“The standard of care for autism is intensive, with early behavioral interventions,” Jacqueline Crawley, co-senior author and the Robert E. Chason Endowed Chair in Translational Research at the MIND Institute, stated in a news release. “In contrast, there are currently no medical treatments that significantly improve the diagnostic symptoms of autism. We are seeking pharmacological targets that correct the biological abnormalities caused by mutations in risk genes for autism.”

A significant number of patients with autism spectrum disorder harbor SHANK3B mutations. Behaviorally, Shank3B knockout mice exhibited repetitive grooming, deficits in aspects of reciprocal social interactions and vocalizations and reduced open field activity, as well as variable deficits in sensory responses, anxiety-related behaviors, learning and memory. Developed at Duke University, Shank3B knockout mice also replicate abnormal brain electroencephalography (EEG) activity.

To better understand this model, the labs at UC Davis and Boston Children’s Hospital compared two independently bred groups of Shank3B knockout mice and control groups. Researchers replicated and extended previously reported behaviors in Shank3B mice, such as repetitive self-grooming and reduced social interaction.

“In each lab, two independently bred cohorts of Shank3B mice and their wildtype littermate controls were tested,” says Crawley. “The behavioral and EEG abnormalities were found to replicate well across the two cohorts in both labs.”

Significant, well-replicated EEG abnormalities and autism-relevant behaviors in Shank3B mice provide an excellent model system for translational evaluation of novel therapeutics for the diagnostic symptoms of autism spectrum disorder, Crawley says. Drug testing requires robust, replicable outcomes of a genetic mutation because preclinical studies are designed to detect whether a drug reverses the abnormalities.

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This is an abridged version of an article we ran in the August 2017 issue of DDNews. To view the full article, click here.